Bacterial polysaccharides suppress induced innate immunity by calcium chelation

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.     

Condition status and parasite infection of Neogobius kessleri and N. melanostomus (Gobiidae) in their native and non-native area of distribution of the Danube River

The success of introduced species is often facilitated by escape from the effects of natural predators and parasites. Introduced species can profit from this favourable situation, attaining higher population densities and greater individual sizes in novel areas. In this study, somatic condition and parasite infection were compared between native and non-native populations of Neogobius kessleri Günther; introduced only within the interconnected Danube and Rhine River system, and N. melanostomus (Pallas); widely introduced throughout several river systems in Europe and North America. Higher values of Fulton’s condition factor were observed in non-native populations of both goby species. Neogobius melanostomus attained higher gonadosomatic index values in non-native populations, indicating potential increased investment in reproduction in its new area. A lower splenosomatic index was observed in non-native populations, especially in N. melanostomus. Parasite infracommunity richness and mean abundance were higher in N. kessleri in both native and non-native populations, suggesting higher susceptibility of N. kessleri to these parasites. Non-native populations of both hosts showed higher infra-community richness as a result of acquiring parasites native to the new area, but lower parasite abundance. Differences in success of the introduction and establishment in new areas between the two fish species may be associated with a relatively low parasite infection rate and a higher gonadosomatic index in non-native populations of N. melanostomus in comparison to N. kessleri.     

Design and synthesis of a simplified inhibitor for XIAP-BIR3 domain

Based on tetrapeptide AVPI, we were able to design and synthesize a new simplified scaffold to inhibit the BIR3 domain of the XIAP protein at low micromolar range. The uncomplicated synthesis and the binding activity of the molecule disclosed here represent an attractive alternative to develop new compounds targeting the protein–protein interaction of XIAP/caspase9.     

Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits

The large-scale production of recombinant biopharmaceutical glycoproteins in the milk of transgenic animals is becoming more widespread. However, in comparison with bacterial, plant cell, or cell culture production systems, little is known about the glycosylation machinery of the mammary gland, and hence on the glycosylation of recombinant glycoproteins produced in transgenic animals. Here the influence is presented of several lactation parameters on the N-glycosylation of recombinant C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed in the milk of transgenic rabbits. Enzymatically released N-glycans of series of rhC1INH samples were fluorescently labeled and fractionated by HPLC. The major N-glycan structures on rhC1INH of pooled rabbit milk were similar to those on native human C1 inhibitor and recombinant human C1 inhibitor produced in transgenic mouse milk, with only the degree of sialylation and core fucosylation being lower. Analyses of individual animals furthermore showed slight interindividual differences; a decrease in the extent of sialylation, core fucosylation, and oligomannose-type glycosylation with the progress of lactation; and a positive correlation between expression level and oligomannose-type N-glycan content. However, when large quantities of rhC1INH were isolated for preclinical and clinical studies, highly consistent N-linked glycan profiles and monosaccharide compositions were found.     

Changes in ponderal index and body mass index across childhood and their associations with fat mass and cardiovascular risk factors at age 15

Background

Little is known about whether associations between childhood adiposity and later adverse cardiovascular health outcomes are driven by tracking of overweight from childhood to adulthood and/or by vascular and metabolic changes from childhood overweight that persist into adulthood. Our objective is to characterise associations between trajectories of adiposity across childhood and a wide range of cardiovascular risk factors measured in adolescence, and explore the extent to which these are mediated by fat mass at age 15.

Methods and Findings

Using data from the Avon Longitudinal Study of Parents and Children, we estimated individual trajectories of ponderal index (PI) from 0–2 years and BMI from 2–10 years using random-effects linear spline models (N = 4601). We explored associations between PI/BMI trajectories and DXA-determined total-body fat-mass and cardiovascular risk factors at 15 years (systolic and diastolic blood pressure, fasting LDL- and HDL-cholesterol, triglycerides, C-reactive protein, glucose, insulin) with and without adjustment for confounders. Changes in PI/BMI during all periods of infancy and childhood were associated with greater DXA-determined fat-mass at age 15. BMI changes in childhood, but not PI changes from 0–2 years, were associated with most cardiovascular risk factors in adolescence; associations tended to be strongest for BMI changes in later childhood (ages 8.5–10), and were largely mediated by fat mass at age 15.

Conclusion

Changes in PI/BMI from 0–10 years were associated with greater fat-mass at age 15. Greater increases in BMI from age 8.5–10 years are most strongly associated with cardiovascular risk factors at age 15, with much of these associations mediated by fat-mass at this age. We found little evidence supporting previous reports that rapid PI changes in infancy are associated with future cardiovascular risk. This study suggests that associations between early overweight and subsequent adverse cardiovascular health are largely due to overweight children tending to remain overweight.     

Human skeletal muscle creatine transporter mRNA and protein expression in healthy,young males and females

The present study investigated whether there were any differences between males and females in respect to creatine transporter (CreaT) gene expression and/or total creatine (TCr) content in human vastus lateralis muscle. Skeletal muscle obtained from young healthy male (n = 13, age: 23.2 ± 5.0 years) and female subjects (n = 12, age: 21.7 ± 4.3 years) was analyzed for CreaT mRNA, CreaT protein and TCr content. Total CreaT protein content in the muscle was similar (p > 0.05) between the sexes. Two bands (~ 55 and 73 kDa) of the CreaT protein were detected in all muscle samples. Both the 55 and the 73 kDa bands were present in similar (p > 0.05) amounts in males compared with females. The 73 kDa band was in greater abundance (p < 0.05) than the 55 kDa band, irrespective of gender. In addition, CreaT mRNA expression relative to -actin mRNA and the TCr content (males: 117.8 ± 2.2, females: 125.3 ± 4.3 mmol.kg^–1 dry mass) were also unaffected (p > 0.05) by gender. These data demonstrate that gender does not influence skeletal muscle TCr content and CreaT gene expression in young human subjects.     

Application of morphometric techniques to postnatal rat testes in organ culture: insights into testis growth

The extent of Sertoli cell proliferation during fetal and neonatal development determines the final adult testis size and potential for sperm output. To gain further knowledge of the factors that regulate Sertoli cell proliferation, the present study used a new approach to analyse changes in morphology and proliferation in the postnatal testis by combining organ culture with morphometric analysis. Fragments of rat testes from days 0 to 10 postpartum were cultured in contact with DMEM for 6 h or 72 h and fixed. The effects of ovine follicle-stimulating hormone (FSH) and activin were studied in an additional 72-h organ culture experiment using day 9 testes. Bromodeoxyuridine (BrdU) was added for the last 6 h of culture to mark proliferating cells. Two-microm sections of the fragments were analysed for morphological changes of the seminiferous cords, and the proportion of BrdU-labelled Sertoli and germ cells was determined. Assessment of 6-h samples revealed growth characteristics consistent with those observed in vivo during days 1-10 of postnatal development. From day 2 onwards, the volume fraction of seminiferous cords began to increase, while significant growth in cross-sectional area of the cords occurred only after day 6. In these culture conditions, germ cell proliferation and testicular architecture was consistent with that expected for the age of the tissue at time of explant. The proportion of dividing Sertoli cells declined from 15-20% at days 0-4 postpartum to below % at day 10 postpartum in the 6-h culture, and it was low or abolished in the 3-day culture at all time points. Activin and FSH together, but not singly, stimulated Sertoli cell proliferation in the 72-h culture. This paper presents a new approach to analysis of in vitro testis development. The combination of fragment culture and stereological analysis permits rigorous and detailed assessment of developmental changes in the postnatal testis.     

Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon

Gill-associated virus (GAV) of the black tiger prawn Penaeus monodon has been implicated as a cause of periodic production losses in Australia since 1996. We report here the development of a real-time quantitative RT-PCR (qRT-PCR) for GAV. A dilution series of in vitro transcribed RNA was used to determine the sensitivity limit of the qRT-PCR and as a standard for GAV quantification. A linear relationship between cycle threshold (Ct) values and input RNA was obtained over a wide concentration range between 4.86 x 10(9) and 0.5 template copies per reaction, the latter being the test detection limit. The qRT-PCR was used to follow the progression of GAV levels in a group of 15 adult male P. monodon with chronic GAV infections that were super-infected by intramuscular injection of an inoculum containing high levels of GAV. By Day 9 post-injection, cumulative mortalities reached 100% (15/15) in the GAV-injected prawns and 40% (2/5) in placebo-injected prawns. Spermatophores were collected at the beginning, and together with other tissues, at the end of the trial. Prawns were also bled at regular intervals to collect circulating haemocytes. The qRT-PCR revealed that GAV loads increased significantly in haemocytes collected from both the control and super-infected prawns (p = 0.010). This increase was significantly higher in the super-infected prawns (p = 0.047). The rapid increase in GAV levels in super-infected P. monodon was expected. However, the increase in the control prawns was not, and indicates that repetitive bleeding and handling stress can stimulate GAV proliferation in chronically infected P. monodon.     

Imidazo[1,2-b]pyridazines: a potent and selective class of cyclin-dependent kinase inhibitors

Modification of imidazo[1,2-a]pyridine CDK inhibitors lead to identification of less lipophilic imidazo[1,2-b]pyridazine series of CDK inhibitors. Although several equivalent compounds from these two series have similar structure and show similar CDK activity, the SAR of the two series differs significantly. Protein inhibitor structure determination has confirmed differences in binding mode and given some understanding of these differences in SAR. Potent and selective imidazo[1,2-b]pyridazine inhibitors of CDK2 have been identified, which show >1 microM plasma levels following a 2mg/kg oral dose to mice.     

Rab9 GTPase regulates late endosome size and requires effector interaction for its stability

Rab9 GTPase resides in a late endosome microdomain together with mannose 6-phosphate receptors (MPRs) and the tail-interacting protein of 47 kDa (TIP47). To explore the importance of Rab9 for microdomain establishment, we depleted the protein from cultured cells. Rab9 depletion decreased late endosome size and reduced the numbers of multilamellar and dense-tubule-containing late endosomes/lysosomes, but not multivesicular endosomes. The remaining late endosomes and lysosomes were more tightly clustered near the nucleus, implicating Rab9 in endosome localization. Cells displayed increased surface MPRs and lysosome-associated membrane protein 1. In addition, cells showed increased MPR synthesis in conjunction with MPR missorting to the lysosome. Surprisingly, Rab9 stability on late endosomes required interaction with TIP47. Rabs are thought of as independent, prenylated entities that reside either on membranes or in cytosol, bound to GDP dissociation inhibitor. These data show that Rab9 stability is strongly influenced by a specific effector interaction. Moreover, Rab9 and the proteins with which it interacts seem critical for the maintenance of specific late endocytic compartments and endosome/lysosome localization.